This R01 renewal project aims to explain the neural origins of breathing behavior at the cellular and synaptic level. It advances understanding of the brainstem pre-Btzinger complex (preBtC), which is acknowledged to be the principal site driving respiration in humans and all terrestrial mammals so far studied. Also, this project examines interneurons of the intermediate reticular formation, adjacent to the preBtC, which may give rise to respiratory premotor neurons. The intellectual driving force for this project is te discovery by the PI's team - and French colleagues - that the key rhythmogenic preBtC interneurons in perinatal mice are derived from embryonic precursors that express transcription factor Dbx1 (i.e., Dbx1 preBtC neurons). This project exploits this new knowledge and by coupling Dbx1 Cre-driver mice with six different flox-STOP reporter strains to perform a spectrum of experiments in vivo and in vitro such as patch-clamp recordings, cell-specific laser ablations with physiological monitoring, and optogenetic manipulations that interrogate network properties. Aim 1 uses juvenile and adult mice (in vivo and in vitro) to examine whether Dbx1 preBtC neurons are rhythmogenic beyond embryonic and neonatal stages of development. Aim 2 uses embryonic and neonatal mice in vitro in conjunction with cell-specific laser ablation methods to test whether preBtC neurons with bursting-pacemaker properties are obligatory for respiratory rhythm generation, offering a fresh approach to a 24-year-old unsolved problem regarding `pacemaker' driven preBtC rhythms. Aim 3 uses perinatal mice in vitro to characterize synaptic interconnections among Dbx1 neurons and quantify the input-output relationship. These experiments elucidate recurrent synaptic excitation in Dbx1 preBtC neurons, which is also putatively rhythmogenic. Aim 4 uses perinatal through adult mice (in vivo and in vitro) to examine whether Dbx1 neurons in the adjacent intermediate reticular formation serve as the first layer of premotor neurons for respiratory movements of the tongue (genioglossus) and pharynx. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project is significant because it characterizes the cellular and synaptic mechanisms that animate the key genetic class of neurons (i.e., Dbx1) at the core of the respiratory oscillator, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who - with French colleagues - first characterized the role of Dbx1 neurons in the preBtC and now is poised to further discover their detailed properties and downstream premotor counterparts. If this project succeeds, neuroscience would finally know the cellular and synaptic origins of a significant central pattern- generating circuit in a mammal and the point of origin for an important behavior, breathing.